Summary. Our overarching research goal is the development of a minimally invasive, receptor-based technology able to monitor the concentration of effectively any specific molecule (irrespective of its chemical reactivity) in the living body. To this end, we have already demonstrated the ability of electrochemical, aptamer-based (E-AB) sensors to monitor a range of drugs and metabolites in situ in the blood vessels and brain of awake, freely moving rats with seconds resolution and measurement durations of hours. A potentially significant limitation of the platform, however, is its reliance on nucleic acid aptamers, the limited chemical complexity of which will no doubt ultimately restrict the number of targets amenable to detection using the approach. In response, we propose here to develop the first sensors in this class that instead employ proteins as their recognition elements. Specifically, we propose two aims that will significantly de-risk the development of protein-folding-based electrochemical sensors, setting the stage for their development as a powerful new approach to molecular monitoring. First, we will expand beyond the single, proof-of-principle example (a sensor fabricated using the FynSH3 domain as our receptor) we have realized to date to sensors against three additional, clinically important targets (the sepsis-diagnostic cytokine IL-6, the chemotherapeutic and immune modulator methotrexate, and the immunosuppressant cyclosporine) as evidence that our design strategy is general. Second, we will adapt these sensors, which we have already shown are capable of multi-hour performance in undiluted whole blood in vitro, to the more challenging measurement conditions found in vivo. If successful, the R21-scale project described here will lay the foundation for an R01-level research program that couples this technology with in vitro and in vivo protein selection to create sensors supporting the continuous, real time measurement of many clinically important molecules, including narrow-therapeutic-index drugs and protein biomarkers indicative of the status of many grievous, rapidly progressive diseases.